Luana Pennacchi

The best approach for early prediction of fetal gender by using free fetal DNA from maternal plasma

Detection of free fetal DNA (ffDNA) in maternal blood during pregnancy has given rise to the possibility of developing new noninvasive approaches for early prenatal diagnosis.

Soluble adhesion molecules: marker of pre-eclampsia and intrauterine growth restriction.

Objective: This study monitored circulating plasma levels of soluble vascular cellular adhesion molecule-1 (sVCAM-1), intracellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) in women with healthy pregnancies, with pregnancy-induced hypertension (PIH), with pre-eclampsia and with pregnancies with isolated intrauterine growth restriction (IUGR) in order to determine whether elevated concentrations have a predictive value for the clinical signs of those pregnancy-induced disorders. Methods: Plasma concentrations of sVCAM-1, sICAM-1 and sE-selectin were determined in healthy pregnant women at each trimester of pregnancy and in pregnant women with PIH, pre-eclampsia and IUGR using commercial kits. Results: In the group of healthy pregnant women, plasma levels of sVCAM-1, sICAM-1 and sE-selectin did not change throughout pregnancy. No significant differences in the levels of these molecules were observed between healthy pregnant women at the third trimester of pregnancy and women with PIH. In addition, concentrations of soluble adhesion molecules were significantly higher in women with pre-eclampsia than in the group of women with healthy pregnancies. Only sVCAM-1 and sE-selectin levels were significantly higher in women with IUGR compared to healthy pregnant women. Conclusions: Abnormally circulating levels of sVCAM-1, sICAM-1 and sE-selectin may have a predictive value for pre-eclampsia and IUGR, as they may be linked with endothelial activation and/or damage.

Prenatal diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies

In the present study, we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities.

A New Methodology of Fetal Stem Cell Isolation, Purification, and Expansion: Preliminary Results for Noninvasive Prenatal Diagnosis

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p < 0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Identification of universal mRNA markers for noninvasive prenatal screening of trisomies

The discovery of placental transcripts in peripheral blood of pregnant women prompted us to investigate which was the most appropriate biological specimen, between plasma and serum, to easily detect them and to exploit hPL (human placental lactogen), βhCG (human chorionic gonadotrophin β‐subunit), LOC90625, and TFPI2 (tissue factor pathway inhibitor 2) levels in order to establish whether an abnormal variation degree of presence of these placental transcripts are likely to be associated to specific fetal trisomies.

Body mass index associated to rs2021966 ENPP1 polymorphism increases the risk for gestational diabetes mellitus

Abstract Gestational diabetes mellitus (GDM) is a condition of impaired glucose tolerance occurring in 1–14% of all pregnancies. This wide range reflects pathological involvement of single nucleotide polymorphisms (SNPs) and maternal weight as risk factors. This study evaluated the association of genetic component and maternal factors to identify women with higher risk of developing GDM. About 240 pregnant women characterized by negative Oral Glucose Tolerance Test (−OGTT) and 38 with positive OGGT (+OGTT) were enrolled. SNPs for ENPP1, NRF1, VEGFA, CEBPA, and PIK3R1 were analyzed by SNP genotyping. An association study was performed and differences in genotype and allele frequencies between cases and controls were analyzed by χ2 test. +OGTT was associated to high values of pre-gestational body mass index (BMI) and age. SNP for ENPP1 gene was associated to +OGTT, while genetic variants for other genes did not correlate to GDM. ENPP1 homozygous for A allele and heterozygous showed altered frequencies in +OGTT when compared with −OGTT. Association of both pre-gestational BMI and age with AA homozygous genotype increased significantly the risk to +OGTT. Our results demonstrate that correlation of age and pre-gestational BMI with homozygous for A allele increased significantly the risk of impaired glucose tolerance and GDM. Chinese abstract 妊娠期糖尿病（GDM）是糖耐量受损的一种状况，妊娠期发病率为1%∼14%。发病率变化范围大是涉及单核苷酸多态性病理与母体体重为危险因素的反映。本研究对遗传组份和母体因素进行评估，从而确定发展为GDM较高风险的妇女。实验中240名妇女口服葡萄糖耐量试验阴性（—OGTT），38名妇女OGTT阳性（+OGTT）。通过SNP基因分型方法对单核苷酸多态性基因组ENPP1、NRF1、VEGF、CEBPA和PIK3R1进行分析，并进行了相关性研究，同时通过χ2检验分析病例组和对照组间基因型和等位基因频率的差异。OGTT阳性与孕前高体重指数和年龄密切相关。SNP中ENPP1基因与OGTT阳性相关，而其他变异基因与GDM无关联。与OGTT阴性相比，OGTT阳性患者中ENPP1纯合子和杂合子A等位基因频率不同。因此孕前体重指数（BMI）、年龄与A等位基因纯合基因型表达可显著增加OCTT阳性的风险。我们的研究结果表明年龄、孕前BMI与A等位基因的纯合型相关，这种相关性可显著增加糖耐量受损和GDM的发病风险。

Non-Invasive Prenatal RHD Genotyping Using Cell-Free Fetal DNA from Maternal Plasma: An Italian Experience.

Background: This study assessed the diagnostic accuracy of a non-invasive approach to fetal RHD genotyping using cell-free fetal DNA in maternal plasma and a combination of methodological strategies. Methods: Real-time PCR (qPCR) was performed on 216 RhD-negative women between weeks 10+0 and 14+6 of gestation (1st qPCR). qPCR was repeated (2nd qPCR) to increase the amount of each sample for analysis, on 95 plasma aliquots that were available from first trimester blood collection (group 1) and on 13 samples that were collected between weeks 18+0 and 25+6 of gestation (group 2). qPCR was specific for exons 5 and 7 of the RHD gene (RHD5 and RHD7). The results were interpreted according to the number of positive replicates of both exons. Results: 1st qPCR: diagnostic accuracy was of 93.3%. Diagnostic accuracy increased from 90.5% (1st qPCR) to 93.7% (2nd qPCR) in group 1 and from 84.6% (1st qPCR) to 92.3% (2nd qPCR) in group 2. These increments were not statistically significant. Conclusion: Our approach to RHD genotyping in early pregnancy yielded high diagnostic accuracy. Increasing the amount of DNA analyzed in each sample did not improve significantly the diagnostic accuracy of the test.

Maternal smoking does not affect the amount of cell-free fetal DNA in maternal plasma during the 1st trimester of pregnancy

Abstract CffDNA, from 344 non-smoking, 38 smoking and 33 ex-smoking pregnant women at 11 + 0–13 + 6 gestational weeks, was extracted and quantified by the multicopy DYS14, as the fetal DNA marker and using the quantitative real-time PCR 7300 detection system. The smoking habit was based on maternal self-report, confirmed by cotinine levels and male fetuses were verified by phenotype at birth. The genders of newborns were compared with DYS14-cffDNA analysis, achieving a 100% diagnostic accuracy of the test. A total of 177 non-smokers, 18 smokers and 22 ex-smoker pregnancies with male fetuses were identified by the cffDNA concentration. Results showed that smoking status was not associated with different amounts of DYS14-cffDNA (p = 0.159), suggesting the possibility of offering cffDNA testing to all pregnant women, even if they are active smokers or ex-smokers, and the test can be unadjusted for smoking status.

Diagnostic accuracy of non‐invasive prenatal sex determination: a large‐scale study

Non-invasive prenatal determination of fetal sex offers a promising alternative to invasive diagnosis of X-linked diseases and fetal disorders of ambiguous genitalia (1–4). Using free-fetal DNA (ffDNA) from maternal plasma (5) and different detection systems for Y chromosome (6–9), researchers achieved high diagnostic accuracy in fetal sex prediction during the first trimester of pregnancy without excluding variable percentage of false positives (FP) and false negatives (FN) (10, 11). We previously showed higher performance of DYS14 than SRY detection system without reaching an optimal diagnostic accuracy because of the low percentage of FN and FP (12). The aim of this study was to enhance the performance of our DYS14 detection system by introducing new key elements, increasing the volume of maternal plasma for DNA extraction and employing innovative interpretation criteria of results. Finally, we audited the overall diagnostic accuracy on a large-scale study to verify whether the new protocol ensured the correct fetal sex determination. We analyzed, in two different phases, plasma samples from 513 women at 10–15 weeks of gestation. During the first non-blinded phase, we established the best threshold value (TV) to discriminate male and female fetuses and, in the second blinded phase, we applied it to assess its diagnostic performance. Fetal sex was verified with the analysis of karyotype or confirmed with phenotype at birth. The study was approved by our Regional Ethical Committee. Genomic DNA was extracted from maternal plasma (500 μl in the first and 1000 μl in the second phase) using QIAmp DSP Virus kit (Qiagen, Hilden, Germany) and analyzed by real-time polymerase chain reaction (PCR) 7300 detection system (Applied Biosystems, Foster City, CA) using DYS14 detection system to measure the quantity of ffDNA (three replicates for each maternal sample) and telomerase reverse transcriptase detection system as a quality control. Real-time PCR [quantitative PCR (qPCR)] reaction was set up as previously described (12). Results were expressed as median values with range for a descriptive statistics and analyzed by using receiver operating characteristic (ROC) curves, calculated by spss software 17.0 (SPSS Inc., Chicago, IL), to set the TV, in terms of ffDNA concentration and number of DYS14positive replicates. Karyotype or phenotype at birth revealed that among the 115 pregnant women analyzed in the first phase of study, 55 delivered one daughter and 60 one son. By evaluation of qPCR results (Table 1), we set our first TV by analysis of ROC curve calculated using the number of DYS14-positive replicates (data not shown). However, the TV was not satisfactory, except for samples with 0, 1 or 2 DYS14-positive replicates that were clearly identified as female fetus. Only one sample from a male pregnancy had 0 DYS14-positive replicates. The critical point was the interpretation of those results with three DYS14-positive replicates, as they occurred in either female or male pregnancies. Two samples from female pregnancy gave three positive replicates but with a very low ffDNA concentration, whereas all the samples from male pregnancy gave three DYS14-positive replicates with elevated ffDNA concentration (Table 1). Therefore, we built a new ROC curve combining ffDNA concentration and the number of DYS14positive replicates and selecting only those samples with three DYS14-positive replicates (2 from female and 59 from male pregnancies). We individuated the best ffDNA concentration value as 1.42 GE/ml as it allowed us to reach a 100.00% diagnostic sensitivity [95% (confidence interval) CI: 93.9–100.0] and 100.00% diagnostic specificity (95% CI: 19.3–100.0) (Fig. 1). In the second phase of study, karyotype or phenotype at birth revealed that 208 delivered a son and 190 a daughter.

The potential usefulness of free fetal DNA in maternal blood for prenatal fetal gender determination in multiple pregnancies.

We applied a noninvasive prenatal test for the determination of fetal gender in multiple pregnancies by using free fetal DNA circulating in maternal blood in order to evaluate whether the quantification of male DNA could distinguish the fetal gender and the number of male and female fetuses in multiple pregnancies. We enrolled consecutively 44 women with twin pregnancies between 11-14 weeks of gestation. Peripheral maternal blood was collected, and genomic DNA was extracted from maternal plasma and analyzed for the multicopy DYS 14 sequence by using real-time PCR to quantify male DNA. Results showed that male DNA concentration was significantly higher in twin pregnancies with at least one male fetus, compared to twin pregnancies with only female fetuses. Comparing male DNA concentration in pregnancies with two male fetuses versus pregnancies with one female fetus and one male fetus, we did not obtain a significant difference between the two groups due to a slight overlapping of the range values. Therefore, our test correctly predicted fetal gender, distinguishing twin pregnancies with at least one male fetus from twin pregnancies with only female fetuses, with a diagnostic accuracy of 100%. For distinguishing pregnancies with two male fetuses from pregnancies with both female and male fetuses, a diagnostic accuracy of 76.1% was achieved.